Gestationally dependent immune organization at the maternal-fetal interface.

The immune system and placenta have a dynamic relationship across gestation to accommodate fetal growth and development. High-resolution characterization of this maternal-fetal interface is necessary to better understand the immunology of pregnancy and its complications. We developed a single-cell framework to simultaneously immuno-phenotype circulating, endovascular, and tissue-resident cells at the maternal-fetal interface throughout gestation, discriminating maternal and fetal contributions. Our data reveal distinct immune profiles across the endovascular and tissue compartments with tractable dynamics throughout gestation that respond to a systemic immune challenge in a gestationally dependent manner. We uncover a significant role for the innate immune system where phagocytes and neutrophils drive temporal organization of the placenta through remarkably diverse populations, including PD-L1+ subsets having compartmental and early gestational bias. Our approach and accompanying datasets provide a resource for additional investigations into gestational immunology and evoke a more significant role for the innate immune system in establishing the microenvironment of early pregnancy.

FLT3 ligand regulates thymic precursor cells and hematopoietic stem cells through interactions with CXCR4 and the marrow niche.

Impaired immune reconstitution after hematopoietic stem cell transplantation (HSCT) is attributed in part to impaired thymopoiesis. Recent data suggest that precursor input may be a point of regulation for the thymus. We hypothesized that administration of FLT3 ligand (FLT3L) would enhance thymopoiesis after adoptive transfer of aged, FLT3L-treated bone marrow (BM) to aged, Lupron-treated hosts by increasing murine HSC (Lin[minus]Sca1+c-Kit+ [LSK] cells) trafficking and survival. In murine models of aged and young hosts, we show that FLT3L enhances thymopoiesis in aged, Lupron-treated hosts through increased survival and export of LSK cells via CXCR4 regulation. In addition, we elucidate an underlying mechanism of FLT3L action on BM LSK cells-FLT3L drives LSK cells into the stromal niche using Hoescht (Ho) dye perimortem. In summary, we show that FLT3L administration leads to: (1) increased LSK cells and early thymocyte progenitor precursors that can enhance thymopoiesis after transplantation and androgen withdrawal, (2) mobilization of LSK cells through downregulation of CXCR4, (3) enhanced BM stem cell survival associated with Bcl-2 upregulation, and (4) BM stem cell enrichment through increased trafficking to the BM niche. Therefore, we show a mechanism by which FLT3L activity on hematopoeitic and thymic progenitor cells may contribute to thymic recovery. These data have potential clinical relevance to enhance thymic reconstitution after cytoreductive therapy.

Development and validation of a cell-based assay system to assess human immunodeficiency virus type 1 integrase multimerization.

Multimerization of HIV-1 integrase (IN) subunits is required for the concerted integration of HIV-1 proviral DNA into the host genome. Thus, the disruption of IN multimerization represents a new avenue for intervening HIV-1 infection. Here, we generated a cell-based assay system to assess IN multimerization using a newly constructed bimolecular fluorescence complementation (BiFC-IN) system. BiFC-IN proteins were efficient in emitting fluorescence, and amino acid (AA) substitutions associated with IN multimerization attenuated fluorescence, suggesting that the BiFC-IN system may be useful for evaluating the profile of IN multimerization. A recently reported non-catalytic site IN inhibitor (NCINI), which allosterically induces IN over-multimerization/aggregation, significantly increased fluorescence in the BiFC-IN system. An IN's substitution, A128T, associated with viral resistance to NCINIs, decreased the NCINI-induced increase of fluorescence, suggesting that A128T reduces the potential for IN over-multimerization. Moreover, E11K and F181T substitutions known to inhibit IN tetramerization also reduced the NCINI-induced fluorescence increase, suggesting that NCINI-induced IN over-multimerization was more likely to occur from tetramer subunits than from dimer subunits. The present study demonstrates that our cell-based BiFC-IN system may be useful in elucidating the profile of IN multimerization, and also help evaluate and identify novel compounds that disrupt IN multimerization in living cells.